Stiffness anisotropy coordinates supracellular contractility driving long-range myotube-ECM alignment

In skeletal muscle tissue, injury-related changes in stiffness activate muscle stem cells through mechanosensitive signaling pathways. Functional muscle tissue regeneration also requires the effective coordination of myoblast proliferation, migration, polarization, differentiation, and fusion across multiple length scales. Here, we demonstrate that substrate stiffness anisotropy coordinates contractility-driven collective cellular dynamics resulting in C2C12 myotube alignment over millimeter-scale distances. When cultured on mechanically anisotropic liquid crystalline polymer networks (LCNs) lacking topographic features that could confer contact guidance, C2C12 myoblasts collectively polarize in the stiffest direction of the substrate. Cellular coordination is amplified through reciprocal cell-ECM dynamics that emerge during fusion, driving global myotube-ECM ordering. Conversely, myotube alignment was restricted to small local domains with no directional preference on mechanically isotropic LCNs of same chemical formulation. These findings reveal a role for stiffness anisotropy in coordinating emergent collective cellular dynamics, with implications for understanding skeletal muscle tissue development and regeneration.